Most cells in adult animals are terminally differentiated, meaning that they cannot normally become cells of a different type. However, certain cells (“multipotential” cells) retain the ability to differentiate (i.e., become cells of one of several types), even in adult tissues.
Bone marrow contains various types of multipotential cells, including precursors of non-hematopoietic cells (e.g., MSCs) and stem cells which can differentiate to become various hematopoietic cells (Friedenstein et al., 1976, Experimental Hematology 4:267-274; Castro-Malaspina et al., 1980, Blood 56:289-301; Mets and Verdonk, 1981, Mech. Aging. Dev. 16:81-89; Piersma et al., 1985, Experimental Hematology 13:237-243; Friedenstein et al., 1987, Cell and Tissue Kinetics 20:263-272; Caplan, 1991, J. Ortho. Res. 9:641-650; Prockop, 1997, Science 276:71-74). Bone marrow cells that are precursors of non-hematopoietic cells and tissues have been designated “plastic-adherent cells” or “colony-forming unit fibroblasts”, because they readily adhere to culture dishes and form fibroblast-like colonies (Piersma et al., 1985, Exp. Hematol. 13:237-243; Owen and Friedenstein, 1988, In: Cell and Molecular Biology of Vertebrate Hard Tissues, pp. 42-60, Ciba Foundation Symposium, Chichester, UK). MSCs are designated mesenchymal stem cells or mesenchymal progenitor cells by still others (Caplan, 1991, J. Ortho. Res. 9:641-650), owing to their ability to differentiate into a variety of non-hematopoietic cells. In addition, these cells have been designated MSCs, because they appear to arise from supporting structures found in marrow and because they can act as feeder layers for the growth of hematopoietic stem cells in culture (Prockop, 1997, Science 276:71-74; Anklesaria, 1987, Proc. Natl. Acad. Sci. USA 84:7681-7685). MSCs have also been used as a feeder layer to obtain cultures of an enriched population of hematopoietic stem cells (Kiefer, 1991, Blood 78:2577-2582).
MSCs have recently attracted renewed interest, because they appear to provide circulating progenitor cells which can repopulate non-hematopoietic tissues (Pereira et al., 1998, Proc. Natl. Acad. Sci. USA 95:1142-1147; Ferrari et al., 1998, Science 279:1456, 1528-1530), and because they can potentially serve as effective vehicles for cell and gene therapy (Caplan, 1991, J. Ortho. Res. 9:641-650; Prockop, 1997, Science 276:71-74).
The original reports of MSCs by Friedenstein et al. (1976, Exp. Hematol. 4:267-274) have been extensively replicated and extended by other investigators (Castro-Malaspina et al., 1980, Blood 56:289-301; Mets and Verdonk, 1981, Mech. Aging Dev. 16:81-89; Piersma et al., 1985, Exp. Hematol. 13:237-243; Howlett et al., 1986, Clin. Ortho. and Related Res. 213:251-263; Anklesaria et al., 1987, Exp. Hematol. 15:636-644; Owen and Friedenstein, 1988, In: Cell and Molecular Biology of Vertebrate Hard Tissues, pp. 42-60, Ciba Foundation Symposium, Chichester, UK; Beresford et al., 1992, J. Cell Sci. 102:341-351; Cheng et al., 1994, Endocrinology 134:277-286; Rickard et al., 1994, Dev. Biol. 161:218-228; Clark and Keating, 1995, Ann. New York Acad. Sci. 770:70-78). The results of those investigations establish that MSCs which are isolated by adherence to tissue culture glass and plastic surfaces are multipotential, in that they can differentiate into osteoblasts, chondrocytes, and adipocytes. Subsequently, MSCs isolated in this manner were demonstrated to differentiate into myoblasts and myotubes (Wakitani et al., 1994, Muscle and Nerve 18: 1417-1426; Prockop, 1997, Science 276:71-74).
Friedenstein et al. (1987, Cell and Tissue Kinetics 20:264-272) demonstrated that MSCs obtained from rabbits can be amplified to achieve 20 to 30 doublings in culture, and that such cells could synthesize bone following implantation in a diffusion chamber in vivo. More recently, Kuznetsov et al. (1997, J. Bone Miner. Res. 12:1335-1347) demonstrated that about 60% of single colony-derived MSCs obtained from human donors form bone when implanted into immuno-deficient mice using implantation vehicles containing a hydroxyapatite and tricalcium phosphate ceramic matrix. Bruder et al. (1997, J. Cell. Biochem. 64:278-294) reported that human MSCs derived from bone marrow aspirates could be induced to undergo 38±4 doublings in culture, and that MSCs were able to differentiate into osteoblasts in vitro following such doublings.
Stromal cells are believed to strongly influence the microenvironment within bone marrow in vivo. When isolated, MSCs are initially quiescent, but eventually begin dividing. Thus, MSCs can be cultured in vitro. MSCs have been used to generate colonies of fibroblastic adipocytic and osteogenic cells, when maintained in culture under appropriate conditions. MSCs can also be made to differentiate into cartilage cells and myoblasts. If plastic- or glass-adherent MSCs are cultured in the presence of hydrocortisone or other selective conditions, cell populations enriched for hematopoietic precursor cells or osteogenic cells can be obtained (Carter et al., 1992, Blood 79:356-364 and Bienzle et al., 1994, Proc. Natl. Acad. Sci. USA, 91:350-354).
The potential of MSCs to differentiate into cells of various lineages in cell culture has been described (e.g. Rickard et al., 1996, J. Bone Miner. Res. 11:312-324; Stanford et al., 1995, J. Biol. Chem. 270:9420-9428; Kuznetsov et al., 1997, J. Bone Miner. Res. 12:1335-1347; Kelly et al., 1998, Endocrinology 139:2622-2628; Lecka-Czernik et al., 1999, J. Cell Biochem. 74:357-371; Uzawa et al., 1999, J. Bone Miner. Res. 14:1272-1280). Others have also described the potential of MSCs and related cells obtained from bone marrow to differentiate into multi-cellular lineages in vivo. Infusion of MSCs into lethally irradiated mice resulted in appearance in the mice of progeny of the MSCs in a variety of tissues, including bone, cartilage, and lung tissues (Pereira et al., 1998, Proc. Natl. Acad. Sci. USA 95:1142-1147). The progeny MSCs expressed a marker type I procollagen gene in bone (a tissue which normally contains type I collagen), but did not express the same gene in collagen (a tissue which does not normally contain type I collagen). Infusion of male MSCs into irradiated female mice led to the presence of Y chromosome-containing cells in primary cultures of fibroblasts derived from a number of different tissues. Nilsson et al. (1999, J. Exp. Med. 189:729-734) demonstrated the presence of donor marked cells as osteoblasts and osteocytes in mice into which large numbers of MSCs were infused, but which did not undergo marrow ablation. Hou et al. (1999, Proc. Natl. Acad. Sci. USA 96:7294-7299) detected donor MSCs as osteoblasts and osteocytes in the bone of lethally irradiated mice. In those MSCs, expression of a marker CAT gene was driven by an osteocalcin promoter.
Engraftment of MSCs or related bone marrow-derived cells into muscle has been reported. Ferrari et al. (1998, Science 279:1456, 1528-1530) reported engraftment of donor MSCs into muscle following either local injection or systemic injection. Gussoni et al., (1999, Nature 401:390-393) examined incorporation into muscle of a rare marrow cell defined as “side population” (SP) cells. SP cells were originally identified in marrow by Goodell et al. (1997, Nat. Med. 12:1337-1345) as rare cells that are small in size and rapidly secrete a series of labeling dyes, because they contain a large amount of a multi-drug-resistant protein. SP cells are CD34-negative, but are precursors of CD34-positive hematopoietic stem cells and other hematopoietic cells. Gussoni et al. infused small numbers of SP cells into lethally irradiated mdx mice that have a mutation in the dystrophin gene. These investigators observed that about 4% of the muscle fibers in the mdx mice contained dystrophin derived from the donor cells. In addition, muscle SP cells could be isolated from skeletal muscle of the mdx mice, and these cells could be used to re-populate marrow and to rescue the mice from marrow ablation.
These and other reports demonstrate that systemic administration of MSCs to marrow-ablated mice leads to appearance of progeny of the MSCs in marrow, lung, liver, bone, cartilage, and muscle tissues. These results indicate that MSCs can differentiate into cells of different lineages, and thus can be used to supplement tissues of various types by systemic or local administration of MSCs. Thus, MSCs (i.e. MSCs obtained from a donor or expanded from a patient's own complement of MSCs) can be used to treat diseases of at least marrow, lung, liver, bone, cartilage, and muscle tissues by replacing or supplementing diseased tissue. Furthermore, recombinantly-constructed MSCs (i.e. harboring an exogenous gene or a normal gene) can be used to deliver a gene product to any of these tissues (e.g. to deliver dystrophin to muscle tissue).
The potential of MSCs and related marrow-derived cells to engraft into the central nervous system has been described. Eglitis et al., (1997, Proc. Natl. Acad. Sci. USA 94:4080-4085) described the presence of donor-derived astrocytes following infusion of whole marrow into recipient immuno-deficient mice. Significant shortcomings of prior attempts to use bone marrow-derived cells to treat central nervous system disorders include that cells infused into nervous system tissues have not behaved appropriately following infusion, and that it has been difficult to obtain cells for infusion.
Azizi et al. (1998, Proc. Natl. Acad. Sci. USA 95:3908-3913) reported that either rat or human MSCs infused into the basal ganglia of adult rats integrated and migrated in a manner similar to paraventricular astrocytes that have many of the properties of neural stem cells. Kopen et al. (1999, Proc. Natl. Acad. Sci. USA 96:10711-10716) demonstrated that murine MSCs infused into the paraventricular region of newborn mice integrated and migrated in a manner similar to neural stem cells. The cells appeared to increase in number as the mouse brains enlarged. Some of the cells differentiated into astrocytes. Other cells appeared in large numbers in neuron-rich regions and may have differentiated into neurons. Use of MSCs for treatment of disorders of the central nervous system has also been described in PCT application publication number WO99/43286. The potential for inter-convertibility of cells between bone marrow and central nervous system tissues was also emphasized by a recent report in which it was demonstrated that neural stem cells can reconstitute the hematopoietic system in mice that have undergone marrow ablation (Bjornson et al., 1999, Science 283:534-537).
Clinical trials involving administration of MSCs and related cells have been carried out. For example, a trial has been initiated in which children afflicted with severe osteogenesis imperfecta (type III) underwent marrow ablation, followed by transfusion of allogeneic marrow from an HLA-compatible brother or sister (Horwitz et al., 1999, Nat. Med. 5:309-313; Horwitz et al., 1999, “Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with severe osteogenesis imperfecta,” Abstracts of the 7th Intl. Meeting on Osteogenesis Imperfecta, Montreal). In this trial, it was hypothesized that whole bone marrow may contain significant numbers of MSCs and related osteoblast precursors that can replace the recipient's osteoblasts and thereby convert a severe form of osteogenesis imperfecta into a milder form of the disease. Five children in the trial exhibited significant improvement, as assessed, for example, by decreased fracture rate and increased total body mineral content. However, donor cells accounted for only 1.3 to 2% of the osteoblasts in three of four patients from whom bone marrow biopsies were obtained. Thus, these results can be interpreted in various ways. For example, it may be that the whole bone marrow transfusions contained only a small number of late progenitors of osteoblasts, and that these cells provide only a temporary improvement in the status of bone in these patients. Other possibilities are that marrow ablation alone produced an undefined benefit, even though bone marrow transplants have, in general, led to osteopenia in children (e.g. Bhatia et al., 1998 Bone Marrow Transplantation 22:87-90).
In another clinical trial, White et al., (1999, “Marrow cell transplantation for infantile hypophosphatasia,” Abstracts of the 7th Intl. Meeting on Osteogenesis Imperfecta, 1999) treated an eight-month-old child afflicted with infantile hypophosphatasia, a genetic deficiency of the alkaline phosphatase that occurs in osteoblasts. The child underwent bone marrow ablation, and then received a bone marrow transplant from an HLA-compatible sister. One hundred days following the transplant, the child demonstrated a remarkable reversal of her worsening skeletal disease and other improvements. However, the beneficial effects were no longer present six months later. The child was then given a non-T-cell depleted boost with MSCs which were expanded from her sister and which were administered without further marrow ablation. Radiographs nine months following the booster dose showed significant improvement that appeared to persist.
Despite the great interest in examining the biology of MSCs and their potential use for therapy, there is still no generally accepted protocol for isolating and expanding MSCs in culture. Most experiments relating to differentiation of MSCs have been performed using cultures of MSCs that have been isolated primarily by virtue of the MSCs tight adherence to tissue culture dishes, as described (Friedenstein et al., 1976, Exp. Hematol. 4:267-274; Friedenstein et al., 1987, Cell Tissue Kinet. 20:263-272). Others have attempted to prepare more homogenous MSC populations (e.g. Long et al., 1995, J. Clin. Invest. 95:881-887; Simmons et al., 1991, Blood 78, 55-62; Waller et al., 1995, Blood 85:2422-2435; Rickard et al., 1996, J. Bone Miner. Res. 11:312-324; Joyner et al., 1997, Bone 21:1-6). However, none of these protocols has gained wide acceptance. In addition, these protocols have been primarily designed to isolate osteoblast precursors. Use of these protocols has not been investigated to determine if they yield cells that are truly multipotential.
Recently, there has been renewed interest in using the Stro-1 antibody to isolate MSCs (e.g. Gronthos et al., 1996, J. Hematotherapy 5:15-23; Byers et al., 1999, J. Pathol. 187:374-381; Steart et al., 1999, J. Bone Miner. Res. 14:1345-1356). However, it may be that Stro-1 primarily stains large and granular cells in cultures of MSCs. Therefore, this antibody may be primarily useful for identifying or isolating only cells that are committed to the osteoblast lineage.
This review of the literature demonstrates that transplantation of MSCs have significant therapeutic and gene transfer uses. However, prior art methods for isolating MSCs and inducing their proliferation have practical limitations, including the extent of population expansion that can be achieved using prior art methods. There remains a critical need for methods of reliably inducing significant proliferation of MSCs in culture without inducing differentiation of the MSCs as they proliferate. The present invention satisfies this need.